First-Line Pembrolizumab Therapy of Non-Small Cell Lung Cancer: Baseline Metabolic Biomarkers Predict Outcomes

Quantitative biomarkers derived from positron-emission tomography/computed tomography (PET/CT) have been suggested as prognostic variables in immunecheckpoint inhibitor (ICI) treated non-small cell lung cancer (NSCLC). As such data for first-line ICI therapy and especially for chemotherapy-ICI combinations are still scarce, we retrospectively evaluated baseline F-FDG-PET/CT of 85 consecutive patients receiving first-line pembrolizumab with chemotherapy (n=70) or as monotherapy (n=15). Maximum and mean standardized uptake value, metabolic tumor volume (MTV), total lesion glycolysis and bone marrow-/ spleen to liver ratio (BLR/SLR) were calculated. Kaplan-Meier analyses and Cox-regression models were used to assess progression-free/overall survival (PFS/OS) and their determinant Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 12 November 2021


Introduction
Positron-emission tomography/computed tomography (PET/CT) enables functional visualization and quantification of malignant lesions in various cancer entities. In lung cancer, the application of PET/CT is recommended in staging of both limited and advanced disease. [1][2][3][4] In the last decade, immune-checkpoint inhibitors (ICI) directed against programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) brought major therapeutic advances especially in non-small cell lung cancer (NSCLC). Originally introduced as second-line therapy, [5][6][7][8] PD-1/PD-L1-blockade alone or as a combination therapy together with platinum-based doublet chemotherapy has moved into first-line therapy, leading to improved survival and response rates. [2,[9][10][11] A considerable fraction of patients has shown a long-term benefit in follow-up, [12,13] although by far not all respond to such treatment.
The challenge of predicting favorable responses is still ongoing, whereas biomarkers like PD-L1 expression, tumor mutational burden or presence of targetable genetic tumor alterations are being widely applied. [14] Also, clinical or laboratory parameters like Eastern Cooperative Oncology Group (ECOG) performance status, neutrophil to lymphocyte ratio or lactate dehydrogenase have been suggested, [14,15] but each of them provides only limited predictive properties on the individual patient's level.
Several biomarkers derived from PET/CT imaging have been reported to predict outcomes in various malignancies treated with ICI. [4,16,17] Concerning NSCLC treated with chemotherapy or ICI, especially volume-based PET/CT variables like total (whole-body) metabolic tumor volume (MTV) and total lesion glycolysis (TLG) have been shown predictive properties in terms of therapy response and survival. [18][19][20][21][22][23][24][25][26] Of interest, the combination of the quantitative PET/CT biomarker MTV and the blood-based biomarker derived neutrophil to lymphocyte ratio (DNLR) Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 12 November 2021 had predictive impact in NSCLC patients receiving ICI. [26][27][28] Similar to DNLR, also PET/CT allows an estimation of the activity of lymphatic tissues, as usually expressed by the bone marrow to liver ratio (BLR) or the spleen to liver ratio (SLR).
In malignant melanoma, higher BLR as well as SLR have been reported to be associated with an unfavorable prognosis. [29,30] Although bone marrow hypermetabolism is a known prognostic factor in resected or chemo(-radio)therapy treated NSCLC, [31][32][33] implications for ICI therapy of advanced NSCLC have not yet been reported. Importantly, with some exceptions, [21,24,25] most existing evidence for quantitative PET/CT biomarkers in the context of ICI therapy is based on mono-immunotherapy cohorts in higher therapy lines, reflecting the initial regulatory approvals for nivolumab, pembrolizumab and atezolizumab. [5][6][7][8] However, first-line ICI therapy in combination with chemotherapy or as monotherapy for tumors with PD-L1 expression ≥50% is currently regarded standard of care. [1,2,10,11,34,35] Whether the existing data on quantitative PET/CT biomarkers can be transferred to the present therapeutic setting is thus questionable, especially due to the increased application of chemotherapy together with ICI.
Consequently, it was our aim to evaluate the clinical implications of quantitative biomarkers derived from pre-therapy 18 F-FDG-PET/CT in a well-characterized retrospective cohort of patients receiving first-line ICI therapy with pembrolizumab in combination with chemotherapy or as monotherapy. 6

Patients:
Eighty-five consecutive patients who had undergone 18  statement. [36] According to institutional standards, patients with PD-L1 expression <50% received a chemo-ICI combination with pembrolizumab and carboplatin/pemetrexed for nonsquamous and carboplatin/paclitaxel for squamous histology. [10,11] Chemotherapy was given for four cycles with no further maintenance therapy, pembrolizumab was continued until progression or toxicity. Patients with PD-L1 expression ≥50% could either receive pembrolizumab monotherapy or a combination with platinum-based doublet chemotherapy. [35] Patients were retrospectively followed from first-line ICI therapy initiation on to death or censored at the date of last verified contact. Disease progression and survival were retrospectively assessed by reviewing the relevant medical records, especially imaging studies and death certificates. First-line therapy was defined as first systemic treatment in stage IV or not otherwise treatable stage III disease, whereas previous therapies in potentially curable stages were not 7 considered. We excluded patients in clinical trials, on ICI/ICI combination therapies and patients, who had previously received ICI for NSCLC or other malignancies.
Image Acquisition Protocol and Analysis: PET/CT imaging was accomplished in the staging process usually two to four weeks before therapy initiation, however a time span of a maximum of three months was allowed for inclusion if no tumor-specific therapy had been applied in that time.
PET/CT scans were performed using a dedicated Siemens Biograph 40 Truepoint PET/CT scanner (Siemens Medical Solutions, Illinois). Patients were kept fasting for at least six hours and blood glucose levels were measured before the injection of 18 F-FDG imaging to ensure that values were below 150 mg/dL. 18 F-FDG was administered at a dose of 3.7 MBq/kg through a peripheral vein sixty minutes prior to imaging. Sequential overlapping emission scans of the neck, chest, abdomen, and pelvis were acquired. PET imaging was performed in 3D mode at three minutes per bed position, using the same axial field as the CT scan. We performed image reconstruction using an ordered subset expectation maximization iterative reconstruction algorithm on a 128x128-pixel matrix (AW-OSEM, 2 iterations, 8 subsets), followed by post-reconstruction filtering using a Gaussian filter applied at 5.0mm Full Width at Half Maximum. All patients had attenuation-corrected images without intravenous contrast agent application. All PET/CT studies were reviewed by two specialist nuclear medicine physicians, who were blinded to the clinical data. For further analysis of quantitative PET/CT biomarkers, imaging data were transferred to a Hermes Workstation (Hermes Medical Solutions, Stockholm, Sweden).
Semiquantitative analysis of 18 F-FDG tumor uptake was performed with the Affinity Software Tool®. In this research, all SUV values were based on body weight. To determine the SUVmax, irregular isocontour regions of interest were drawn over 8 abnormal findings at 50% of maximum pixel value within the lesion. The volumes of all segmented individual lesions were summed to obtain the whole-body MTV for each patient. [37] TLG was calculated as the product of the MTV and the SUVmean within the MTV. [38] BLR and SLR were calculated as the ratio of bone marrow/spleen and liver SUVmax. Bone marrow SUVmax was measured in the vertebral bodies of L1-L5, whereas areas with vertebral fractures and tumors/metastases were omitted. Spleen and liver SUVmax were calculated in a spherical VOI of three cm in the respective organ in an area with physiological morphology in the CT images, excluding e.g., metastases.
Laboratory Analyses: C-reactive protein (CRP) and LDH were assessed using a Cobas® 8000 modular analyzer (Roche Diagnostics International AG, Rotkreuz, Switzerland), lymphocyte count was analyzed using a Sysmex® XN-3000 hematology analyzer (Sysmex Europe GmbH, Norderstedt, Germany). Expression of PD-L1 on tumor cells was determined using a 22C3 assay for Autostainer Link 48 by Dako (Agilent Technologies, Santa Clara, CA), a negative PD-L1 status was defined as membranous staining on <1% of viable tumor cells.
Response Assessment: Radiological response to ICI therapy was routinely assessed every six to nine weeks by a CT scan of the chest and the upper abdomen using iodinated contrast medium unless contraindicated. Re-staging could be preponed due to suspected disease progression and additional imaging modalities like cerebral magnetic resonance tomography could be conducted according to the treating clinician's judgement.

Response was graded according to the Response Evaluation Criteria in Solid Tumors
(RECIST), version 1.1. [39] Disease control rate (DCR) was defined as patients with 9 complete/partial remission (CR/PR) or stable disease (SD) versus those with progressive disease (PD). Patients, who had died before the first scheduled CT restaging (n=11) were counted as PD.

Conclusions
Quantitative baseline PET/CT biomarkers in ICI-treated advanced NSCLC patients can provide essential predictive biomarker information, both concerning metabolic tumor characteristics, but also reflecting the immune system. The combination of high MTV and BLR identifies a clinically highly relevant group of patients with a poor prognosis that warrants intensified diagnostic and therapeutic efforts by the clinician as well as future research activity concerning additional treatment options.
Author Contributions: All authors have made significant contributions to this manuscript and meet the criteria for authorship as established by the International Committee of Medical Journal Editors, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising, or critically reviewing the article. All authors gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.